Recombinant antibodies and their fragments have become key to the design of high affinity specific targeting drugs. Recent construction of engineered targeting molecules has demonstrated the need for use of minimal binding fragments that are rebuilt into multivalent high affinity reagents. Antibody fragment units have also been found with a range of molecules for gene therapy, imaging, immunotherapy, radiotherapy, chemotherapy and pro-drug therapy. ScFv (30 kD) are usually the smallest antibody fragment that retains specific binding characteristics. ScFv are produced by randomly connecting the variable heavy (VH) and variable light (VL) chain immunoglobulin genes together using a biologically inert flexible linker. While scFv molecules have been produced from existing monoclonal antibodies, phage display libraries now provide a multitude of scFv from a single source, allowing those with optimal binding characteristics to be simultaneously selected along with the genes encoding the displayed scFv (1. Pavlinkova et al. (1999) J Nucl Med, 40:1536–1546; Viti et al. (1999) Cancer Res, 59: 347–352; Winter et al. (1994)Annu. Rev. Immunol, 12: 433–455; Clackson et al. (1991) Nature, 352: 624–628; Hoogenboom et al. (1998) Immunotechnology, 4: 1–20; Phage display of peptides and proteins: a laboratory manual. San Diego: Academic Press, 1996).
One of the epithelial mucin family of molecules, MUC-1 has received considerable interest as an antigen target because it is widely expressed on a large number of epithelial cancers and is aberrantly glycosylated making it structurally and antigenically distinct from that expressed by non-malignant cells (Barratt-Boyes (1996) Cancer Immunol Immunother, 43: 142–151; Price et al. (1998) Tumor Biology, 19: 1–20; Peterson et al. (1991), Pages 55–68 In: Breast Epithelial Antigens, R. L. Ceriani. (ed.), New York: Plenum Press). The dominant form of MUC-1 is a high molecular weight molecule comprised of a large highly immunogenic extracellular mucin-like domain with a large number of 20 amino acid tandem repeats, a transmembrane region, and a cytoplasmic tail (Quin et al. (2000) Int J Cancer, 87:499–506; McGucken et al. (1995) Human Pathology, 26: 432–439; Dong et al. (1997) J. Pathology, 183: 311–317). In normal epithelial tissue, MUC-1 is localized to the apical region of the cells; malignant transformation results in upregulation of MUC-1 by gene amplification and/or increased transcriptional activation and the distribution of MUC-1 on the cell surface is no longer confined to the apical region (Bieche and Lidereau (1997) Cancer Genetics and Cytogenetics, 98: 75–80). While the function of MUC-1 still awaits clarification, high cytoplasmic expression of MUC-1 expression have been associated with poor prognosis in patients with breast and/or ovarian cancers. MUC-1 has also been demonstrated to play a role in cell adhesion, cell signaling and immune responses (Quin et al. (2000) Int J Cancer, 87:499–506; McGucken et al. (1995) Human Pathology, 26: 432–439; Dong et al. (1997) J. Pathology, 183: 311–317; Henderson et al. (1998) J Immunother, 21: 247–256).
A substantial number of anti-MUC-1 monoclonal antibodies (MoAb) have been produced with the majority of these MoAb recognizing epitopes contained within the twenty amino acid tandem repeat that are bordered on each side by a serine and a threonine (Barratt-Boyes (1996) Cancer Immunol Immunother, 43: 142–151.; Price et al. (1998) Tumor Biology, 19: 1–20; Peterson et al. (1991), Pages 55–68 In: Breast Epithelial Antigens, R. L. Ceriani. (ed.), New York: Plenum Press; Fontenot et al. (1993) Cancer Res, 53: 5386–5394 Pemberton et al. (1996) J Biol. Chem., 271: 2332–2340; Regimbald et al. (1996) Cancer Res, 56: 4244–4249; Kotera et al. (1994) Cancer Res, 54: 2856–2860). These anti-MUC-1 MoAbs have been used primarily as diagnostic agents to identify tumors and monitor levels of circulating antigen. A few MUC-1 MoAbs have been used to deliver targeted radiation to tumors as radioimmunotherapy. In ovarian cancer, the HMFG1 antibody was used to deliver high dose yttrium to the peritoneum in patients with minimal residual disease after receiving chemotherapy (Papadimitriou et al. (1999) Biochimica et. Biophysica Acta, 1455: 301–313; Maraveyas et al. (1994) Cancer, 73: 1067–1075). A clear survival benefit was demonstrated when compared to historical controls (Id.). The results were significant enough to prompt a phase III multicenter trial for treatment of ovarian cancer. Another anti-MUC-1 MoAb (BrE-3) labeled with 90Y has also been used in the treatment of breast cancer (Papadimitriou et al. (1999) Biochimica et. Biophysica Acta, 1455: 301–313; DeNardo et al. (1997) J Nucl Med, 38: 1180–1185; Kramer et al. (1998) Clin Cancer Res, 4: 1679–1688; Press et al. (1993) N Engl J Med, 329: 1219–1224). Transient clinical response warrants further studies (Papadimitriou et al. (1999) Biochimica et. Biophysica Acta, 1455: 301–313; DeNardo et al. (1997) J Nucl Med, 38: 1180–1185; Kramer et al. (1998) Clin Cancer Res, 4: 1679–1688; Press et al. (1993) N Engl J Med, 329: 1219–1224; DeNardo et al. (1991) Int J Rad Appl Instrum [B]. 18: 621–631; Stewart and Brunjes (1993) Brain Res. 628: 243–253).
While radioimmunotherapy using intact MoAbs has been utilized in the treatment of breast cancer and other solid tumors, therapeutic success has been limited by the large size of the MoAb (150 kD) inhibiting blood clearance and retarding accumulation of the radiopharmaceutical at the tumor site(s) (Maziere et al. (1986) Exp Cell Res, 167: 257–261).